“Secondary metabolites” was originally used by plant physiologists to classify botanicals (e.g. dyes, fragrances, and medicinals) with no obvious function in the plants that produced them. It now encompasses a heterogous group of compounds, usually of low relative molecular mass, and made mostly but not exclusively by organisms without a nervous system (i.e., bacteria, fungi, and plants). The notion of secondary metabolism was embrassed by microbiologists in the 1960s, with attention focusing on antibiotics and other bioactive microbial products (Bently, R. et al, Annu. Rev. Microbio. (1999) 53: 411-446).
It is well known that members of the genus Streptomyces produce a great many antibiotics and other classes of biologically active secondary metabolites. The genus Streptomyces belongs to the order Actinomycetales. In general, the order Actinomycetales means Actinomycetes. Actinomycetes make over 60% of the known secondary metabolites that are produced by microorganisms, and amongst them nearly 80% are made by members of the genus Streptomyces, with other genera trailing numerically (Kieser, T. et al, Practical Streptomyces Genetics (2000): 10-11).
Most antibiotics applied in clinical were first isolated from the metabolites of microbes including bacteria, fungi and actinomycetes, in which Streptomyces are known to be the most effective producers. Genetic recombination and manipulation in Streptomyces have been established by D. A. Hopwood and his co-workers (Hopwood et al. Genetic manipulation of Streptomyces, a laboratory manual, 1985). So far, treatment with mutagens and the screening of resultant clones has been repeated and adopted as a procedure for the improvement of antibiotic-producing Streptomyces and produced good results. However, this method has some disadvantageous properties such as labor-consuming, time-consuming, costly, non-reproductive and low frequency etc. The current intentional methods for improving strains are genetic recombination and manipulation that represent an important technique in strain improvement such as protoplast fusion, structural gene amplification, regulatory genes and resistance genes cloning etc. (Ikeda et al. Actinomycetologica 1991, 5:86-99). Obviously, these methods require a better knowledge about biochemistry and genetics of antibiotic production.
As described above, some methods for improving strains have been invented and applied in fermentation industry. It is known that a productivity of actinorhodin in Streptomyces coelicolor can be improved by conferring streptomycin resistance to it (Protein, Nucleic Acid, Enzyme, vol. 44, No. 13, p 1967-1974 (1999); Kagaku to Seibutsu, vol. 37, No. 11, p 731-737 (1999)). However, no report has ever been made concerning more effective methods to increase the productivity of the secondary metabolites.
An object of the present invention is to provide a method for increasing the productivity of the secondary metabolite by a microorganism in a labor-saving, time-saving, high efficient and semi-rational way and being applicable for the strains without more knowledge of, for example, antibiotic biochemistry and genetics.